Summary Proprotein convertase subtilisin kexin type 9 (PCSK9) is a circulatory protein that binds to the low-density lipoprotein receptor (LDLR), induces its degradation, and increases LDL-cholesterol (LDL-C) levels. Thus, PCSK9 is a therapeutic target to increase LDLR expression and reduce plasma LDL-C levels, and PCSK9 inhibitors have recently been approved for use in the US and Europe. Plasma PCSK9 levels are highly correlated to LDL-C levels, both because elevated PCSK9 levels increase plasma LDL levels by decreasing LDLR and because up to 40% of plasma PCSK9 is physically associated with LDL, a discovery initially made in our laboratory and later confirmed by others. Plasma PCSK9 is found in two main forms, an intact form (62 kDa) and a furin-cleaved form (55 kDa). We show that PCSK9 associated with LDL is mainly in the intact 62- kDa form, whereas the rest of plasma PCSK9 is mainly as the 55 kDa furin-cleaved fragment. The intact, LDL- associated PCSK9 seems to have an increased capacity to bind and degrade LDLR. Our preliminary results also suggest that LDL protects PCSK9 from cleavage by furin. However, it is still unclear what drives the compartmentalization of the molecular forms of circulating PCSK9, and whether there are functional correlates to this compartmentalization. Thus, we propose studies to clarify the connection between plasma PCSK9 forms, molecular drivers of their lipoprotein association, and LDLR degradation activity. Moreover, we propose to develop a high-throughput method to detect LDL-bound PCSK9 in plasma and to study PCSK9 association with other apoB-containing lipoproteins, such as Lp(a), and with intracellular apoB. Finally, we will study the mechanisms leading to the unexpectedly large time delay between the initial PCSK9-LDLR contact and the eventual degradation of the LDLR, as this seems to be due to a complex intracellular routing of PCSK9 which includes a re-secretion/recycling step. The central hypothesis of this proposal is that LDL carries a significant portion of the intact plasma PCSK9 form (62 kDa), which may behave differently in LDLR binding and degradation compared with the other major form of plasma PCSK9 (55 kDa). The results from these studies will enhance our understanding of both the physiologic role of PCSK9 association with lipoproteins and the mechanism by which PCSK9 inhibition produces therapeutic gains, and may inform alternative strategies to inhibit the effect of PCSK9 on LDLR through interruption of PCSK9 association with lipoproteins. .